This project will develop a new generation of paramagnetic, functionally oriented probes for the needs of electron paramagnetic resonance (EPR) spectroscopy and imaging, particularly for in vivo applications. EPR- based techniques are far from attaining their maximum potential, predominantly because of a lack of stable in vivo exogenous spin probes available. All the advantages of application of wide classes of nitroxide probes to biomedicine are largely wiped out by their rapid degradation in tissues to EPR-silent products. In this project several new approaches will be used to develop paramagnetic probes with increased in vivo stability based on the original idea of the construction of the nano-Sized Particles with the Incorporated Nitroxides, or nanoSPINs. These nanoSPINs, being permeable to small analytes will separate sensing nitroxides from biological reductants. The nanoSPIN sensors will be used to detect physiologically important species, namely H+ (pH) and nitric oxide (NO). This will fill a niche between fluorescent probes, which have advanced our detection capabilities at cellular and subcellular levels, and NMR/MRI, which have provided spectroscopic and imaging capabilities in intact living animals and humans. However, NMR/MRI suffers from the lack of sensitivity (1000 fold or lower than EPR) and specificity. The specific aims are: (SA1) Development of effective methods for the nanoSPIN design. The proposed strategies are based on two matrixes for nitroxide encapsulation, sol gel "glasses" and phospholipid bilayer vesicles, including use of polymerized liposomes. The small ion permeability of the liposomes will be ensured by incorporation of "pore formers" such as gramicidin A. (SA2) Physicochemical characterization of pH- and NO-sensitive nanoSPINs. Quantitative characterization of the nanoSPINs, particularly functional sensitivity and stability in biological tissues, is absolutely crucial, both for the optimization of the preparation procedures and for efficiency of their applications. (SA3) To study the role of myocardial acidosis and NO generation in ischemic hearts and in the model of ischemic preconditioning using developed nanoSPINs. We hypothesize that alterations in pH homeostasis and NO production play critically important roles in ischemic preconditioning (IPC). To test the hypothesis, myocardium acidosis and NO production will be monitored noninvasively by EPR in ischemic control and preconditioned hearts. (SA4) To apply in vivo EPR measurements of pH and NO generation in models of mouse heart regional ischemia reperfusion with ischemic preconditioning. In order to test our pH and NO hypothesis in IPC, we will use this in vivo mouse heart model to noninvasively monitor the variations of myocardial pH and NO generation and their correlations to the protective mechanisms of IPC using developed nanoSPINs. The results may provide an opportunity for the design of corresponding therapeutic approaches. In summary, the success of this project may have a significant impact on the future of in vivo EPR spectroscopy and bioimaging applications to medicine. PUBLIC HEALTH RELEVANCE: This project will develop a new generation of paramagnetic functionally oriented probes, termed nanoSPINs, for the needs of electron paramagnetic resonance (EPR) spectroscopy and bioimaging, particularly for in vivo applications to medicine. Application experiments will use pH- and NO-sensitive nanoSPINs in isolated rat hearts and in vivo in a {models of mouse heart regional ischemia reperfusion with ischemic preconditioning} and will provide new opportunities for designing corresponding therapeutic approaches.